Ectopic activation of the Wnt signaling pathway leads to increased cellular growth and division in experimental organisms and mutations in Wnt pathway genes are tightly linked to the genesis of certain cancers in humans (Polakis P., Genetics & Development, 9:15-21(1999)). Signaling through the Wnt pathway begins with a Wnt ligand, one of a family of at least 16 members in mammals. (Cadigan K. M., et al., Genes & Development, 11:3286-3305 (1997)). Wnts are secreted growth factors that signal through cell surface Frizzled (Fz) transmembrane receptors to initiate the signal cascade (Bhanot P., et al., Nature, 282:225-30 (1996); Yang-Snyder J, et al., Current Biol., 6:1302-06 (1996); and He X, et al., Science, 275:1652-54 (1997)).
Members of the Dishevelled (Dsh) family are activated upon ligand binding to Fz (Klingensmith J, et al., Genes Dev., 8:118-30 (1994)) causing inhibition of glycogen synthase kinase-3β (GSK-3β, Zw3, shaggy). Inhibition of GSK-3β activity prevents phosphorylation of β-catenin (Armadillo, Arm) thus blocking APC- and Axin-mediated degradation of β-catenin. Stabilized β-catenin accumulates and binds to members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of HMG-box transcription factors in the nucleus (Behrens J, et al., Nature, 382:638-42 (1996); Van de Wetering M., et al., Cell, 88:789-99 (1997); Riese J., et al., Cell, 88:777-87 (1997) and Brannon M, et al., Genes Dev., 11:2359-70 (1997) leading to changes in the transcription of growth regulatory genes.
Targets of Wnt-LEF/TCF-regulated transcription include the protooncogene myc (He T. C., et al., Science, 281:1509-12 (1998)), cyclooxygenase-2 (COX-2) (Howe L R, et al., Cancer Res., 59:1572-77 (1999)), Matrilysin/MMP-7 (Crawford H. C., et al., Oncogene, 18:2883-91 (1999) and Brabletz T., et al., Am J Pathol., 155:1033-38 (1999), cyclin D1 (Testu O., et al., Nature, 398:422-26 (1999) and Stutman M., et al., PNAS, 96:5522-27 (1999) and a member of the LEF/TCF family, TCF1 (Roose J., et al., Science, 285:1923-26 (1999)). Activation of the Wnt pathway also affects the expression of members of another signaling family of molecules, e.g. BMP6 (Bone Morphogenetic Protein), a member of the transforming growth factor (TGF)-β superfamily (Raid M., et al., Int J Cancer, 83:38-44 (1999)).
The role of Wnt signaling in cancer was suggested by the discovery that ectopic expression of mouse Wnt1 (int1) caused the formation of mammary tumors in mice (Nusse R., et al., Cell, 31:99-109 (1982)). Recognition that the APC (adenomatous polyposis coli) tumor suppressor gene functions as a component of the Wnt pathway further implicated Wnt signaling in cancer, particularly colon cancer (Bienz M., et al., Cell, 103:311-20 (2000)). APC mutations (or mutations in β-catenin or axin) are found in up to 85% of sporadic forms of colon cancers (Miyoshi Y., et al., Hum Mol Genet., 1:229-33 (1992) and Potter J. D., J Nat'l Cancer Inst., 91:916-32 (1999)).
Studies in model organisms suggest that the expression of the Wnt genes is controlled by a complex network that employs both positive and negative feedback (Brook W. J., et al., Science, 273: 1373-77 (1996) and Theisen H., et al., Development, 122:3939-48 (1996)). If true in man, loss of heterozygosity or mutational change of certain members of the Wnt signaling pathway, e.g. APC or β-catenin, might be expected to alter the expression of upstream genes such as those for pathway ligands or receptors. Vider et al. reported (Vider B-Z, et al., Oncogene, 12:153-58 (1996)) that Wnt2 is expressed at low levels in normal colon but is overexpressed in tumor tissue samples and that Wnt5a was expressed in both nontumorous as well as colonic tumor tissue, though the methodology used did not distinguish between changes in cancer cells themselves and cells derived from surrounding tissues.
The differential expression of downstream components of the Wnt signaling pathway, specifically members of the LEF/TCF family, has also been suggested. For example, TCF4 is normally expressed in colonic mucosa and cancer (Korinek V., et al., Science, 275:1784-87 (1997)) TCF1 is overexpressed in colon cancer cell lines (Meyer K., et al., Int J Cancer, 72:625-30 (1997)).